1. Field of the Invention
The present invention relates to a gene containing a DNA composition having the nucleotide base sequence coding for a 27-kdal protein of Bacillus thuringiensis with insecticidal properties, microorganisms transformed with such DNA, insecticidal compositions containing the protein or transformed microorganisms and to use of said compositions for control of insects, especially Diptera in the larval form.
2. Description of the Prior Art
The gram-positive bacterium Bacillus thuringiensis var. israelensis (Goldberg, L. J. and Margalitt, J. (1977) Mosquito News, 37 355-358) produces a cytolytic protein delta-endotoxin that is lethal to the larvae of mosquitoes and blackfly (diptera) (De Barjac, H (1978) C.R. Acad. Sci. Paris, ser. D 286, 797-800; Thomas, W. E. and Ellar, D. J. (1983) J. Cell Sci., 60 181-197). This delta-endotoxin is synthesized during sporulation as part of a parasporal crystalline protein inclusion (Somerville, H. J. (1978), Trends Biochem. Sci., 108-110; Bulla, L. A., Jr., Bechtel, D. B., Kramer, K. J., Shethna, Y., Aronson, A. I. and Fitz-James, P. C. (1980) CRC Crit. Rev. Microb., 8, 147-204). A combination of the potent activity of this israelensis delta-endotoxin against dipteran disease vectors, with the fact that protein delta-endotoxins produced by other serotypes of B. thuringiensis are highly toxic to a wide range of lepidopteran pest insects (Luthy, P. (1980) FEMS Microb. Lett., 8, 1-7), has provoked considerable scientific and commercial interest in these bacteria over the past 30 years.
Recent reports of the isolation and expression of the delta-endotoxin gene of B. thuringiensis var. kurstaki (Schnepf, H.E. and Whiteley, H. R. (1981) Proc. Natl. Acad. Sci. USA, 78, 2893-2897; Held, G. A., Bulla, L. A., Jr., Ferrari, E., Hoch, J., Aronson, A. I. and Minnich, S. A. (1982) Proc. Natl. Acad. Sci. USA, 79, 6065-6069) and var. berliner (Klier, A., Fargette, F., Ribier, J. and Rapoport, G. (1982) EMBO J., 1, 791-799) indicate that the delta-endotoxin gene may be variously located on plasmid DNA, chromosomal DNA, or both (Schnepf, H. E. and Whiteley, H. R. (1981) Proc. Natl. Acad. Sci. USA, 78, 2893-2897; Held, G. A., Bulla, L. A., Jr., Ferrari, E., Hoch, J., Aronson, A. I. and Minnich, S. A. (1982) Proc. Natl. Acad. Sci. USA, 79, 6065-6069; Klier, A., Fargette, F., Ribier, J. and Rapoport, G. (1982) EMBO J., . 1, 791-799; Kronstad, J. W., Schnepf, H. E. and Whiteley, H. R. (1983) J. Bacteriol., 154, 419-428; Gonzalez, J., Jr., Brown, B. J. and Carlton, B. C. (1982) Proc. Natl. Acad. Sci. USA, 79, 6951-6955). In the case of B. thuringiensis var. israelensis, two independent studies of strains cured of one or more plasmids concluded that delta-endotoxin synthesis is critically dependent on the presence of a 72-75 Md (ca 110 kb) plasmid (Ward, E. S. and Ellar, D. J. (1983) FEBS Lett., 158, 45-49; Gonzalez, J. M., Jr. and Carlton, B. C. (1984) Plasmid, 11, 28-38). Using the newly discovered capacity of B. thuringiensis for plasmid transfer by a conjugation-like mechanism, authors in Gonzalez, J., Jr., Brown, B. J. and Carlton, B. C. (1982) Proc. Natl. Acad. Sci. USA, 79, 6951-6955) have shown that one or more plasmids in several B. thuringiensis strains code for the delta-endotoxin structural gene. However, to date, transfer of plasmids from B. thuringiensis var. israelensis to other B. thuringiensis serotypes has not been achieved (Gonzalez, J., Jr., Brown, B. J. and Carlton, B. C. (1982) Proc. Natl. Acad. Sci. USA, 79, 6951-6955) and it therefore remains possible that the 72-75 Md plasmid encodes a regulator of delta-endotoxin production, rather than the structural gene.
We describe the resolution of this question by the isolation from hhe purified 72-75 Md plasmid of a DNA fragment that includes the israelensis toxin gene that encodes for a protein toxin of about 27,340 Daltons, the complete nucleotide base sequence of the gene and the amino acid sequence of the protein expressed by the gene code and deduced from the nucleotide base sequence of the structural coding segment of the gene.